Dietary Fat and HDL Metabolism. A great unanswered question in nutrition and cardiovascular disease (CVD) is how to interpret the increase in HDL cholesterol concentration that occurs when dietary unsaturated fat is increased and carbohydrate or protein is reduced. This apparent advantage of unsaturated fat is one reason why some experts favor high unsaturated fat diets compared to those with carbohydrate or protein. Other experts are not so confident that that a therapeutic intervention that raises HDL concentration invariably protects against atherosclerosis. There is a sense of discomfort using simple changes in HDL cholesterol for nutrition and health policy. Lack of clarity of the underlying physiology interferes with informed opinion. There are several metabolic pathways that can sustain a high HDL concentration. It is unclear how dietary fat affects them. The critical issue is whether dietary fat improves the steps in HDL metabolism that are involved in reverse cholesterol transport that protects against atherosclerosis. HDL is indisputably a necessary component of the cholesterol transport and homeostatic system, and a low HDL concentration is one of the strongest predictors of high risk of CVD. Low HDL cholesterol is prominent in overweight and obesity, insulin resistance, type 2 diabetes, and metabolic syndrome. This low HDL concentration is associated with accelerated clearance from the blood circulation of HDL particles, measured by their principal protein, apolipoprotein A-I, and a preponderance of small HDL. This suggests a block in the maturation of HDL from small cholesterol-depleted to large cholesterol ester-rich particles, and impaired reverse cholesterol transport. We do not know whether dietary unsaturated fat corrects this dysfunctional metabolism of HDL. We propose a clinical intervention study in 20 people who have low HDL cholesterol concentrations in the typical setting of overweight or obesity, and high plasma triglycerides to determine how dietary unsaturated fat increases their HDL concentration. We will use stable isotopic tracers to label endogenously apolipoprotein A-I, the defining protein of HDL, to trace its metabolism from small nascent particles (pre-beta HDL) to large, cholesterol-loaded mature particles (alpha 1,2, and 3 HDL), and evaluate the extent of the reverse process representing selective removal of cholesterol ester by the liver. We will study whether dietary fat reduces the involvement of apolipoprotein C-III in HDL metabolism. HDL that has apoC-III has an adverse association with CVD, opposite to the major HDL type that does not have apoC-III. This information will prove invaluable in interpreting the established HDL-raising effect of dietary fat in terms of protection against atherosclerosis.